A chromatographic and spectrophotometric study of the products of the reaction of osmium tetroxide with unsaturated lipids.

Thin films of methyl oleate, oleic acid, and di-octadecenoyl phosphatidylcholine were reacted with a 2% solution of OsO(4) in water for 1 hr at 0 degrees . As controls, methyl 9,10-dihydroxystearate and 9,10-dihydroxystearic acid were reacted with OsO(4) in 0.25 N NaOH in methanol for 1 hr at room temperature. The reaction products were isolated, purified, and analyzed by thin-layer chromatography, gas-liquid chromatography, and infrared and visible spectroscopy. In all cases, the products were identified as diesters of osmic acid in which two molecules of fatty acids are linked through 1 molecule of osmic acid.

Isolation and partial characterization of a 110-kD dimer actin-binding protein.

Two Triton-insoluble fractions were isolated from Acanthamoeba castellanii. The major non-membrane proteins in both fractions were actin (30-40%), myosin II (4-9%), myosin I (1-5%), and a 55-kD polypeptide (10%). The 55-kD polypeptide did not react with antibodies against tubulins from turkey brain, paramecium, or yeast. All of these proteins were much more concentrated in the Triton-insoluble fractions than in the whole homogenate or soluble supernatant. The 55-kD polypeptide was extracted with 0.3 M NaCl, fractionated by ammonium sulfate, and purified to near homogeneity by DEAE-cellulose and hydroxyapatite chromatography. The purified protein had a molecular mass of 110 kD and appeared to be a homodimer by isoelectric focusing. The 110-kD dimer bound to F-actin with a maximal binding stoichiometry of 0.5 mol/mol of actin (1 mol of 55-kD subunit/mol of actin). Although the 110-kD protein enhanced the sedimentation of F-actin, it did not affect the low shear viscosity of F-actin solutions nor was bundling of F-actin observed by electron microscopy. The 110-kD dimer protein inhibited the actin-activated Mg2+-ATPase activities of Acanthamoeba myosin I and myosin II in a concentration-dependent manner. By indirect immunofluorescence, the 110-kD protein was found to be localized in the peripheral cytoplasm near the plasma membrane which is also enriched in F-actin filaments and myosin I.

The fine structure of Acanthamoeba castellanii. I. The trophozoite.

The fine structure of the trophozoite of Acanthamoeba castellanii (Neff strain) has been studied. Locomotor pseudopods, spikelike "acanthopodia," and microprojections from the cell surface are all formed by hyaline cytoplasm, which excludes formed elements of the cell and contains a fine fibrillar material. Golgi complex, smooth and rough forms of endoplasmic reticulum, digestive vacuoles, mitochondria, and the water-expulsion vesicle (contractile vacuole) are described. A canicular system opening into the water-expulsion vesicle contains tubules about 600 A in diameter that are lined with a filamentous material. The tubules are continuous with unlined vesicles or ampullae of larger diameter. Centrioles were not observed, but cytoplasmic microtubules radiate from a dense material similar to centriolar satellites and are frequently centered in the Golgi complex. Cytoplasmic reserve materials include both lipid and glycogen, each of which amounts to about 10% of the dry weight.

The fine structure of Acanthamoeba castellanii (Neff strain). II. Encystment.

Encysting cells of Acanthamoeba castellanii, Neff strain, have been examined with the electron microscope. The wall structure and cytoplasmic changes during encystment are described. The cyst wall is composed of two major layers: a laminar, fibrous exocyst with a variable amount of matrix material, and an endocyst of fine fibrils in a granular matrix. The two layers are normally separated by a space except where they form opercula in the center of ostioles (exits for excysting amebae). An additional amorphous layer is probably present between the wall and the protoplast in the mature cyst. Early in encystment the Golgi complex is enlarged and contains a densely staining material that appears to contribute to wall formation. Vacuoles containing cytoplasmic debris (autolysosomes) are present in encysting cells and the contents of some of the vacuoles are deposited in the developing cyst wall. Lamellate bodies develop in the mitochondria and appear in the cytoplasm. Several changes are associated with the mitochondrial intracristate granule. The nucleus releases small buds into the cytoplasm, and the nucleolus decreases to less than half its original volume. The cytoplasm increases in electron density and its volume is reduced by about 80%. The water expulsion vesicle is the only cellular compartment without dense content in the mature cyst. The volume fractions of lipid droplets, Golgi complex, mitochondria, digestive vacuoles, and autolysosomes have been determined at different stages of encystment by stereological analysis of electron micrographs. By chemical analyses, dry weight, protein, phospholipid, and glycogen are lower and neutral lipid is higher in the mature cyst than in the trophozoite.

Interaction of phospholipid vesicles with cells. Endocytosis and fusion as alternate mechanisms for the uptake of lipid-soluble and water-soluble molecules.

Depending on their phospholipid composition, liposomes are endocytosed by, or fuse with, the plasma membrane, of Acanthamoeba castellanii. Unilamellar egg lecithin vesicles are endocytosed by amoeba at 28 degrees C with equal uptake of the phospholipid bilayer and the contents of the internal aqueous space of the vesicles. Uptake is inhibited almost completely by incubation at 4 degrees C or in the presence of dinitrophenol. After uptake at 28 degrees C, the vesicle phospholipid can be visualized by electron microscope autoradiography within cytoplasmic vacuoles. In contrast, uptake of unilamellar dipalmitoyl lecithin vesicles and multilamellar dipalmitoyl lecithin liposomes is only partially inhibited at 4 degrees C, by dinitrophenol and by prior fixation of the amoebae with glutaraldehyde, each of which inhibits pinocytosis. Vesicle contents are taken up only about 40% as well as the phospholipid bilayer. Electron micrographs are compatible with the interpretation that dipalmitoyl lecithin vesicles fuse with the amoeba plasma membrane, adding their phospholipid to the cell surface, while their contents enter the cell cytoplasm. Dimyristoyl lecithin vesicles behave like egg lecithin vesicles while distearoyl lecithin vesicles behave like dipalmitoyl lecithin vesicles.

Differential localization of Acanthamoeba myosin I isoforms.

Acanthamoeba myosins IA and IB were localized by immunofluorescence and immunoelectron microscopy in vegetative and phagocytosing cells and the total cell contents of myosins IA, IB, and IC were quantified by immunoprecipitation. The quantitative distributions of the three myosin I isoforms were then calculated from these data and the previously determined localization of myosin IC. Myosin IA occurs almost exclusively in the cytoplasm, where it accounts for approximately 50% of the total myosin I, in the cortex beneath phagocytic cups and in association with small cytoplasmic vesicles. Myosin IB is the predominant isoform associated with the plasma membrane, large vacuole membranes and phagocytic membranes and accounts for almost half of the total myosin I in the cytoplasm. Myosin IC accounts for a significant fraction of the total myosin I associated with the plasma membrane and large vacuole membranes and is the only myosin I isoform associated with the contractile vacuole membrane. These data suggest that myosin IA may function in cytoplasmic vesicle transport and myosin I-mediated cortical contraction, myosin IB in pseudopod extension and phagocytosis, and myosin IC in contractile vacuole function. In addition, endogenous and exogenously added myosins IA and IB appeared to be associated with the cytoplasmic surface of different subpopulations of purified plasma membranes implying that the different myosin I isoforms are targeted to specific membrane domains through a mechanism that involves more than the affinity of the myosins for anionic phospholipids.

Phagocytosis of latex beads by Acanthamoeba. II. Electron microscopic study of the initial events.

Electron microscopic studies confirm and extend the conclusions derived previously from a quantitative biochemical study of the phagocytosis of polystyrene and polyvinyltoluene latex beads by Acanthamoeba (1). Latex beads 1.305, 1.90, and 2.68 micro in diameter are ingested individually, with each bead tightly surrounded by a membrane derived from the plasma membrane. Latex beads 0.557, 0.264, 0.126, and 0.088 micro in diameter are accumulated at the surface of the ameba and then phagocytosed, with many beads tightly packed within one membrane-bounded vesicle.

Phagocytosis of latex beads by Acahamoeba castellanii (Neff). 3. Isolation of the phagocytic vesicles and their membranes.

A method is described for the rapid and efficient isolation of phagocytic vesicles from large scale cultures of Acanthamoeba castellanii (Neff) that have been incubated with polystyrene latex beads. Cells were allowed to phagocytose latex beads for 30 min and then were homogenized, and the phagocytic vesicles were isolated by one centrifugation through several layers of sucrose. Identity and purity of the phagocytic vesicles were determined by electron microscopy, chemical analyses, and assays of acid phosphatase, alpha- and beta-glucosidase, and reduced nicotinamide adenine dinucleotide dehydrogenase. When phagocytosis was allowed to occur for longer periods the phagocytic vesicles appeared to fuse with each other and perhaps with digestive vacuoles. The resultant vesicles which contained many beads were heavier than those which consisted of only one bead or a few beads with a closely applied membrane. Ultrasonication ruptured the isolated vesicles, and the membranes could then be isolated in 30-50% yield based on phospholipid analysis. These membranes were essentially free of acid hydrolases and, presumably, other soluble proteins, as was also indicated by their low ratio of protein to phospholipid. The membranes have been prepared both as closed vesicles and as open sheets.

Localization of myosin IC and myosin II in Acanthamoeba castellanii by indirect immunofluorescence and immunogold electron microscopy.

Polyclonal antisera have been raised against purified Acanthamoeba myosin II and to a synthetic 26 amino acid peptide that corresponds in sequence to the phosphorylation site of Acanthamoeba myosin IC. These antisera are specific for their respective antigens as determined by immunoblotting after SDS-PAGE of total cell lysates. By using the antisera, localization studies were performed by indirect immunofluorescence and by immunogold electron microscopy. Myosin II occurred in the cell cytoplasm and appeared to be concentrated in the cortex. Immunogold cytochemistry revealed at high resolution that myosin II is organized into rodlike filaments approximately 200 nm long. The antibody raised against the myosin IC synthetic peptide recognized both the plasma membrane and the membrane of the contractile vacuole. The plasma membrane staining was labile to treatment with saponin suggesting an intimate association of the myosin IC with membrane phospholipids. Immunogold cytochemistry with the antimyosin IC synthetic peptide showed that the myosin IC is closely associated with the membrane bilayer.

Plasma membrane association of Acanthamoeba myosin I.

Myosin I accounted for approximately 2% of the protein of highly purified plasma membranes, which represents about a tenfold enrichment over its concentration in the total cell homogenate. This localization is consistent with immunofluorescence analysis of cells that shows myosin I at or near the plasma membrane as well as diffusely distributed in the cytoplasm with no apparent association with cytoplasmic organelles or vesicles identifiable at the level of light microscopy. Myosin II was not detected in the purified plasma membrane fraction. Although actin was present in about a tenfold molar excess relative to myosin I, several lines of evidence suggest that the principal linkage of myosin I with the plasma membrane is not through F-actin: (a) KI extracted much more actin than myosin I from the plasma membrane fraction; (b) higher ionic strength was required to solubilize the membrane-bound myosin I than to dissociate a complex of purified myosin I and F-actin; and (c) added purified myosin I bound to KI-extracted plasma membranes in a saturable manner with maximum binding four- to fivefold greater than the actin content and with much greater affinity than for pure F-actin (apparent KD of 30-50 nM vs. 10-40 microM in 0.1 M KCl plus 2 mM MgATP). Thus, neither the MgATP-sensitive actin-binding site in the NH2-terminal end of the myosin I heavy chain nor the MgATP-insensitive actin-binding site in the COOH-terminal end of the heavy chain appeared to be the principal mechanism of binding of myosin I to plasma membranes through F-actin. Furthermore, the MgATP-sensitive actin-binding site of membrane-bound myosin I was still available to bind added F-actin. However, the MgATP-insensitive actin-binding site appeared to be unable to bind added F-actin, suggesting that the membrane-binding site is near enough to this site to block sterically its interaction with actin.

Quantification and localization of phosphorylated myosin I isoforms in Acanthamoeba castellanii.

The actin-activated Mg(2+)-ATPase activities of the three myosin I isoforms in Acanthamoeba castellanii are significantly expressed only after phosphorylation of a single site in the myosin I heavy chain. Synthetic phosphorylated and unphosphorylated peptides corresponding to the phosphorylation site sequences, which differ for the three myosin I isoforms, were used to raise isoform-specific antibodies that recognized only the phosphorylated myosin I or the total myosin I isoform (phosphorylated and unphosphorylated), respectively. With these antisera, the amounts of total and phosphorylated isoform were quantified, the phosphomyosin I isoforms localized, and the compartmental distribution of the phosphomyosin isoforms determined. Myosin IA, which was almost entirely in the actin-rich cortex, was 70-100% phosphorylated and particularly enriched under phagocytic cups. Myosins IB and IC were predominantly associated with plasma membranes and large vacuole membranes, where they were only 10-20% phosphorylated, whereas cytoplasmic myosins IB and IC, like cytoplasmic myosin IA, were mostly phosphorylated (60-100%). Moreover, phosphomyosin IB was concentrated in actively motile regions of the plasma membrane. More than 20-fold more phosphomyosin IC and 10-fold more F-actin were associated with the membranes of contracting contractile vacuoles (CV) than of filling CVs. As the total amount of CV-associated myosin IC remained constant, it must be phosphorylated at the start of CV contraction. These data extend previous proposals for the specific functions of myosin I isozymes in Acanthamoeba (Baines, I.C., H. Brzeska, and E.D. Korn. 1992. J. Cell Biol. 119: 1193-1203): phosphomyosin IA in phagocytosis, phosphomyosin IB in phagocytosis and pinocytosis, and phosphomyosin IC in contraction of the CV.

Lipids of Acanthamoeba castellanii. Composition and effects of phagocytosis on incorporation of radioactive precursors.

The lipids of Acanthamoeba castellanii (Neff) consist of 52% neutral lipids and 48% polar lipids. Triglycerides account for 75% and free sterols for 17% of the neutral lipids. The major phospholipids are phosphatidylcholine (45%), phosphatidylethanolamine (33%), phosphatidylserine (10%), a phosphoinositide (6%), and diphosphatidylglycerol (4%). The phosphoinositide is unique in that it contains fatty acids, aldehyde, inositol, and phosphate in the ratio of 1.4:0.5:1.1, but it contains no glycerol. Sphingomyelin, cerebrosides, psychosine, and glycoglycerides were not detected, but small amounts of unidentified long chain bases and sugars are present. The rates of uptake of palmitate-1-(14)C and of its incorporation into glycerides and phospholipids were not affected by the phagocytosis of polystyrene latex beads. Although phagocytosis usually decreased the uptake by amebas of phosphate-(32)P, serine-U-(14)C, and inositol-2-(3)H, their subsequent incroporation into phospholipids was not demonstrably stimulated or inhibited by phagocytosis. Phagocytosis did seem to increase the incorporation into ameba phospholipids of phosphatidylcholine-1 ,2-(14)C but not that of phosphatidylethanolamine-1 ,2-(14)C. These experiments, in which the incorporation of radioactive precursors into total cell lipids was measured, do not, of course, eliminate the possibility that localized effects may occur.

Complete nucleotide sequence and deduced polypeptide sequence of a nonmuscle myosin heavy chain gene from Acanthamoeba: evidence of a hinge in the rodlike tail.

We have completely sequenced a gene encoding the heavy chain of myosin II, a nonmuscle myosin from the soil ameba Acanthamoeba castellanii. The gene spans 6 kb, is split by three small introns, and encodes a 1,509-residue heavy chain polypeptide. The positions of the three introns are largely conserved relative to characterized vertebrate and invertebrate muscle myosin genes. The deduced myosin II globular head amino acid sequence shows a high degree of similarity with the globular head sequences of the rat embryonic skeletal muscle and nematode unc 54 muscle myosins. By contrast, there is no unique way to align the deduced myosin II rod amino acid sequence with the rod sequence of these muscle myosins. Nevertheless, the periodicities of hydrophobic and charged residues in the myosin II rod sequence, which dictate the coiled-coil structure of the rod and its associations within the myosin filament, are very similar to those of the muscle myosins. We conclude that this ameba nonmuscle myosin shares with the muscle myosins of vertebrates and invertebrates an ancestral heavy chain gene. The low level of direct sequence similarity between the rod sequences of myosin II and muscle myosins probably reflects a general tolerance for residue changes in the rod domain (as long as the periodicities of hydrophobic and charged residues are largely maintained), the relative evolutionary "ages" of these myosins, and specific differences between the filament properties of myosin II and muscle myosins. Finally, sequence analysis and electron microscopy reveal the presence within the myosin II rodlike tail of a well-defined hinge region where sharp bending can occur. We speculate that this hinge may play a key role in mediating the effect of heavy chain phosphorylation on enzymatic activity.

Plasma and phagosome membranes of Acanthamoeba castellanii.

Plasma membranes were isolated from the ameba Acanthamoeba castellanii by low-speed velocity centrifugation followed by equilibrium centrifugation in a sucrose gradient. The isolated membranes had a high ratio of sterol to phospholipid (0.98 moles/mole) and of phospholipid to protein (0.43 mg/mg). The plasma membranes had very low concentrations of DNA, RNA, lipid inositol, and glycerides. Glycolipids and glycoproteins were enriched in the plasma membranes relative to their concentrations in the whole cell. The plasma membranes were also judged to be of high purity by the absence, or very low level, of enzymatic activities considered to be indicative of other cell membranes, and by electron microscope examination. Alkaline phosphatase and 5'-nucleotidase activities were enriched in the plasma membranes 13-fold relative to the whole homogenate and had higher specific activities in the plasma membranes than in any other cell fractions. A Mg(++) adenosine triphosphatase (ATPase) was enriched sixfold in the plasma membranes relative to the whole homogenate. The phospholipids of the plasma membranes contained more phosphatidylethanolamine and phosphatidylserine and less phosphatidylcholine than did the phospholipids of the whole cells. There were differences in the fatty acid compositions of corresponding phospholipids in the plasma membranes and whole cells but no difference in the ratios of total saturated to unsaturated fatty acids. The membranes of phagosomes isolated from amebae that had ingested polystyrene latex had essentially the same phospholipid, sterol, and enzymatic composition as plasma membranes.

Immunolocalization of myosin I heavy chain kinase in Acanthamoeba castellanii and binding of purified kinase to isolated plasma membranes.

The actin-activated Mg(2+)-ATPase activities of Acanthamoeba myosins I are known to be maximally expressed only when a single threonine (myosin IA) or serine (myosins IB and IC) is phosphorylated by myosin I heavy chain kinase. The purified kinase is highly activated by autophosphorylation and the rate of autophosphorylation is greatly enhanced by the presence of acidic phospholipids. In this paper, we show by immunofluorescence and immunoelectron microscopy of permeabilized cells that myosin I heavy chain kinase is highly concentrated, but not exclusively, at the plasma membrane. Judged by their electrophoretic mobilities, kinase associated with purified plasma membranes may differ from the cytoplasmic kinase, possibly in the extent of its phosphorylation. Purified kinase binds to highly purified plasma membranes with an apparent KD of approximately 17 nM and a capacity of approximately 0.8 nmol/mg of plasma membrane protein, values that are similar to the affinity and capacity of plasma membranes for myosins I. Binding of kinase to membranes is inhibited by elevated ionic strength and by extensive autophosphorylation but not by substrate-level concentrations of ATP. Membrane-bound kinase autophosphorylates to a lesser extent than free kinase and does not dissociate from the membranes after autophosphorylation. The co-localization of myosin I heavy chain kinase and myosin I at the plasma membrane is of interest in relation to the possible functions of myosin I especially as phospholipids increase kinase activity.

Myosin-I nomenclature.

We suggest that the vertebrate myosin-I field adopt a common nomenclature system based on the names adopted by the Human Genome Organization (HUGO). At present, the myosin-I nomenclature is very confusing; not only are several systems in use, but several different genes have been given the same name. Despite their faults, we believe that the names adopted by the HUGO nomenclature group for genome annotation are the best compromise, and we recommend universal adoption.

Structure of biological membranes.

The combined x-ray diffraction and electron microscopic examination of myelin has provided reasonable, but not conclusive, support for its structure as a basically bimolecular leaflet of phospholipid that is partially interspersed with protein. But there is very little basis for extending this concept to biological membranes in general. There is no adequate experimental support for the specific orientation of phospholipids as proposed in the unit membrane theory or for the proposed polar nature of protein-lipid bonds, even in myelin. Membranes differ widely in chemical composition, metabolism, function, enzymatic composition, and even in their electron microscopic image. The only similarity is their general resemblance in electron micrographs, but, until more is known about the chemistry of electron microscopy, this evidence cannot be interpreted with confidence. One positive conclusion to which I have come is that much more chemical evidence must, and can, be obtained. Techniques for the isolation of membranes are improving and protein and lipid chemistry are now highly refined arts. Quantitative analysis of many different membranes is possible and the data can be related in some instances, notably bacterial plasma membranes, to calculations of surface area. Chemical and physical changes induced in membranes of widely different lipid composition by the preparatory procedures of electron microscopy can be determined directly and correlated with the electron microscopic image. Model systems can be assembled whose compositions closely resemble those of biological membranes. Membranes can be disassociated into subunits whose properties can be studied. In particular, x-ray diffraction analysis and electron microscopy by negative staining of reaggregates of lipoproteins isolated from membranes would be very informative. Perhaps most important, the problem of membrane structure must be considered in relation to the problems of membrane function and membrane biosynthesis.

Actin polymerization and ATP hydrolysis.

F-actin is the major component of muscle thin filaments and, more generally, of the microfilaments of the dynamic, multifunctional cytoskeletal systems of nonmuscle eukaryotic cells. Polymeric F-actin is formed by reversible noncovalent self-association of monomeric G-actin. To understand the dynamics of microfilament systems in cells, the dynamics of polymerization of pure actin must be understood. The following model has emerged from recent work. During the polymerization process, adenosine 5'-triphosphate (ATP) that is bound to G-actin is hydrolyzed to adenosine 5'-diphosphate (ADP) that is bound to F-actin. The hydrolysis reaction occurs on the F-actin subsequent to the polymerization reaction in two steps: cleavage of ATP followed by the slower release of inorganic phosphate (Pi). As a result, at high rates of filament growth a transient cap of ATP-actin subunits exists at the ends of elongating filaments, and at steady state a stabilizing cap of ADP.Pi-actin subunits exists at the barbed ends of filaments. Cleavage of ATP results in a highly stable filament with bound ADP.Pi, and release of Pi destabilizes the filament. Thus these two steps of the hydrolytic reaction provide potential mechanisms for regulating the monomer-polymer transition.

Structure and Function of the Plasma Membrane : A biochemical perspective.

The paucimolecular unit membrane model of the structure of the plasma membrane is critically reviewed in relation to current knowledge of the chemical and enzymatic composition of isolated plasma membranes, the properties of phospholipids, the chemistry of fixation for electron microscopy, the conformation of membrane proteins, the nature of the lipid-protein bonds in membranes, and possible mechanisms of transmembrane transport and membrane biosynthesis. It is concluded that the classical models, although not disproven, are not well supported by, and are difficult to reconcile with, the data now available. On the other hand, although a model based on lipoprotein subunits is, from a biochemical perspective, an attractive alternative, it too is far from proven. Many of the questions may be resolved by studies of membrane function and membrane biosynthesis rather than by a direct attack on membrane structure.